Bacillus subtilis controls mycotoxigenic coffee pathogens by volatile-mediated long-range chemical signaling and contact-dependent lipopeptide antagonism.

In this work, the genome of Bacillus subtilis 34a, a strain isolated from healthy coffee plants, was sequenced and analyzed by genome mining to predict its antifungal potential. These predictions were subsequently evaluated by characterizing the volatile and diffusible chemical profiles associated with interactions between B. subtilis 34a and fungal pathogens (Fusarium spp. and Umbelopsis sp.). Dual co-cultivation assays revealed variable susceptibility among fungal strains to the antagonistic activity of Bacillus subtilis 34a, with radial growth inhibition ranging from 15.2% to 54.1%. Ambient ionization mass spectrometry (LTP-MS) analysis identified a conserved bacterial VOC profile consistently produced across all eight interactions, while direct liquid injection (DLI-ESI-MS) confirmed the production of the diffusible metabolites consistent with biosynthetic gene cluster (BGC) predictions, including lipopeptides and iron-competition compounds. Differential analysis revealed marked chemical remodeling during co-culture, characterized by the reduction of fungal mycotoxin-associated signals, including beauvericins, fumonisins, and enniatins, together with the induction of bacterial lipopeptides, particularly surfactins. In addition, interaction-potentiated metabolites, including cyclosporin-like compounds and NRPS-derived lipopeptide homologs, were enriched exclusively during co-culture, indicating the emergence of chemical signatures associated with fungal-bacterial confrontation. The magnitude and composition of these changes varied substantially among fungal strains, indicating pathogen-specific chemical responses during antagonism. These findings demonstrate that the biocontrol activity of B. subtilis 34a involves dynamic, pathogen-specific chemical interactions, providing molecular insights for the rational selection of biocontrol strategies in coffee cultivation. Because B. subtilis holds qualified presumption of safety status, strain 34a is a promising candidate for this application.